Methods and applications for constructing an animal model of herpes zoster neuralgia using recombinant VZV gE protein

Abstract

The application discloses a method for constructing a herpes zoster neuralgia animal model by using a recombinant VZV gE protein and application thereof, and comprises the following steps: preparing an immunogen by using an effective dose of a recombinant VZV gE protein, wherein the recombinant VZV gE protein is expressed by E. coli and obtained by purification; and injecting the immunogen into a non-human mammal in vivo, and inducing a persistent pain behavior in the non-human mammal, so that a herpes zoster neuralgia animal model is constructed. The application has the advantages of high biological safety, accurate simulation, stable model phenotype and good repeatability.

Description

Technical Field

[0001] This invention relates to methods for constructing animal models in the field of biomedical technology, and particularly to a method and application for constructing an animal model of herpes zoster neuralgia using recombinant VZV gE protein. Background Technology

[0002] Postherpetic neuralgia (PHN) is the most common chronic complication following varicella-zoster virus (VZV) infection, with a clinical incidence rate of 9%-34%. Its core characteristic is severe neuropathic pain persisting for more than 3 months after skin lesions have healed, significantly impacting patients' quality of life. Currently, there are no effective preventative and treatment methods for PHN. Elucidating the pathological mechanisms of PHN and developing highly effective therapeutic drugs have become important research directions in the biomedical field. Constructing animal models that accurately mimic the key pathological features and clinical phenotypes of human PHN is the core foundation and crucial prerequisite for conducting related mechanistic research and drug development.

[0003] Currently, the mainstream animal models of herpes simplex virus (PHN) are mainly divided into two categories: VZV live virus inoculation models and HSV-1 alternative models. However, both models have significant drawbacks, which seriously restrict the in-depth advancement and translation of PHN-related research results. Among them, the VZV live virus inoculation model can induce pain behaviors similar to PHN, such as mechanohyperalgesia, in rodents, but it has inherent technical and application limitations: VZV has strict human tropism and cannot effectively infect and replicate in non-human mammals; the pathological process of the model deviates fundamentally from that of human PHN. At the same time, the handling, culture, and experimentation of live viruses must be carried out in high-level biosafety laboratories, which not only significantly increases research costs but also limits the widespread application of this model in basic research. While the HSV-1 alternative model can induce shingles-like skin lesions and pain behaviors in mice through infection, and has a relatively low experimental threshold, HSV-1 and VZV belong to different viral types. Their pathogenic mechanisms, infection pathways, and the immune responses they elicit are significantly different. The research results obtained from this model have extremely low correlation with the pathological mechanism of PHN caused by human VZV infection, making it difficult to translate into clinical applications and unable to provide reliable experimental evidence for PHN mechanism research and drug development.

[0004] In summary, existing PHN animal models cannot simultaneously ensure biosafety, accuracy of pathological simulation, and experimental operability. Developing a safe, accurate, and stable method for constructing PHN animal models has become an urgent need to address the current bottlenecks in PHN research. Summary of the Invention

[0005] The purpose of this invention is to provide a method and application for constructing an animal model of herpes zoster neuralgia using recombinant VZV gE protein. This invention has the advantages of high biosafety, accurate simulation, stable model phenotype, and good reproducibility.

[0006] The technical solution of the present invention: A method for constructing an animal model of herpes zoster neuralgia using recombinant VZV gE protein, comprising the following steps:

[0007] An effective dose of recombinant VZV gE protein was prepared as an immunogen; the recombinant VZV gE protein was expressed in Escherichia coli and obtained by purification.

[0008] The immunogen was injected into a non-human mammal, inducing persistent pain behavior in the non-human mammal, thereby constructing an animal model of herpes zoster neuralgia.

[0009] The above-described method for constructing an animal model of herpes zoster neuralgia using recombinant VZV gE protein, wherein the non-human mammal is a mouse.

[0010] The aforementioned method for constructing an animal model of herpes zoster neuralgia using recombinant VZV gE protein, wherein the concentration of recombinant VZV gE protein in the immunogen is from 0.05 ng / μl to 0.5 ng / μl.

[0011] In the aforementioned method for constructing an animal model of herpes zoster neuralgia using recombinant VZV gE protein, the single injection dose of the immunogen is 30-50 μl.

[0012] The aforementioned method for constructing an animal model of herpes zoster neuralgia using recombinant VZV gE protein involves a subcutaneous injection into the sole of the foot.

[0013] The aforementioned method for constructing an animal model of herpes zoster neuralgia using recombinant VZV gE protein confirms the successful establishment of the animal model by detecting pain-related behaviors in animals through behavioral methods and by examining nerve damage in animals through immunofluorescence and transmission electron microscopy.

[0014] The behavioral methods include one or more of mechanical hyperalgesia, thermal hyperalgesia, and cold hyperalgesia.

[0015] An animal model of herpes zoster neuralgia constructed using the aforementioned method.

[0016] The aforementioned animal model of herpes zoster neuralgia is used in screening or evaluating drugs for the prevention or treatment of herpes zoster neuralgia.

[0017] The aforementioned animal model of herpes zoster neuralgia was used in the study of the pathogenesis of herpes zoster neuralgia.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1. This invention does not use live VZV virus, thus avoiding uncontrollable biological processes such as virus replication and latency. It does not require operation in a high-level biosafety laboratory, significantly reducing biosafety risks and research costs, and therefore has the advantage of high safety.

[0020] 2. This invention directly uses recombinant VZV gE protein, which is related to human pathogenicity, as a pain causative agent to construct a neuroimmune inflammation and pain process driven by VZV antigen specificity. This eliminates the interference of other viruses such as HSV-1, and the model has a higher correlation with human PHN, thus having the advantage of accurate simulation.

[0021] 3. By controlling the dosage and injection site of viral proteins, this invention can induce significant and sustained pain behaviors such as mechanical hyperalgesia, thermal hyperalgesia, and cold hyperalgesia. The model has a high success rate and good reproducibility.

[0022] 4. The model of this invention provides a safe, reliable and effective tool for studying the neuroimmune mechanism of PHN, discovering new drug targets, and screening and evaluating analgesics, and has wide applications. Attached Figure Description

[0023] Figure 1 The image shows the results of a mechanical pain hypersensitivity test after a single injection of recombinant VZV gE protein into the sole of the foot of a male von Frey experimental rat.

[0024] Figure 2 The image shows the results of thermal pain hypersensitivity detection in male rats after a single injection of recombinant VZV gE protein into the sole of their paw using a thermal radiation instrument (Hargreaves method).

[0025] Figure 3 The image shows the results of a single injection of recombinant VZV gE protein into the sole of the foot of a male rat in an acetone test, resulting in cold pain hypersensitivity.

[0026] Figure 4 The image shows the changes in the myelin sheath structure of nerve fibers in the main trunk of the sciatic nerve after a single injection of recombinant VZV gE protein into the sole of the rat's foot. The upper image is a low-power microscopic observation of the nerve tissue (the scale bar corresponds to a larger field of view), and the lower image is a transmission electron microscopic observation of the nerve fibers (the scale bar is 2 μm, showing the ultrastructure).

[0027] Figure 5Colocalization of ATF3, a neuronal injury marker, with NF200 neurons in DRG tissue 2 and 4 weeks after a single injection of recombinant VZV gE protein into the rat paw.

[0028] Figure 6 Colocalization of ATF3, a neuronal injury marker, with CGRP neurons in DRG tissue 2 and 4 weeks after a single injection of recombinant VZV gE protein into the rat paw.

[0029] Figure 7 Colocalization of ATF3 and IB4 neurons, neuronal injury markers, in DRG tissues 2 and 4 weeks after a single injection of recombinant VZV gE protein into the rat paw.

[0030] Figure 8 Figure 1 shows the expression level and morphological results of the myelin marker PRX in the skin tissue of the rat foot 2 and 4 weeks after a single injection of recombinant VZV gE protein into the foot.

[0031] Figure 9 Figure 2 and 4 weeks after a single injection of recombinant VZV gE protein into the sole of the rat paw to show the expression level and morphological results of the myelin marker PRX in the DRG tissue.

[0032] Figure 10 shows the results of related tests on the plantar skin of rats 6 hours, 2 weeks and 4 weeks after a single injection of recombinant VZV gE protein. (a) shows the statistical results of the morphology of nerve fiber marker PGP9.5 and intraepidermal nerve fiber density (IENF), and (b) shows the co-localization results of nerve fiber marker PGP9.5 and VZV gE protein.

[0033] Figure 11 Detailed results of colocalization of PGP9.5, a broad-spectrum nerve fiber marker, with VZV gE protein in rats 6 hours, 2 weeks, and 4 weeks after a single injection of recombinant VZV gE protein.

[0034] Figure 12 KEGG enrichment analysis of rat foot skin tissue transcriptome sequencing 3 weeks after a single injection of recombinant VZV gE protein into the foot.

[0035] Figure 13 KEGG enrichment analysis of DRG tissue transcriptome sequencing 3 weeks after a single injection of recombinant VZV gE protein into the rat paw. Detailed Implementation

[0036] The present invention will be further described below with reference to the accompanying drawings and embodiments, but this should not be construed as limiting the present invention.

[0037] Example 1: Establishment and experiment of a rat model of herpes zoster neuralgia.

[0038] I. Experimental Materials;

[0039] 1. Experimental animals: Male SD rats, each weighing 180g, were selected and used for experiments after one week of acclimatization. The breeding environment met the standard for routine animal experiments.

[0040] 2. Reagents related to recombinant VZV gE protein: recombinant VZV gE protein (ab43050), cell culture grade PBS buffer.

[0041] 3. Experimental instruments: biosafety cabinet, cell culture incubator, 100kD cutoff tangential flow ultrafiltration system, sucrose density gradient centrifuge, ion exchange chromatography equipment, metal bath, microsyringe (specification adapted to 40μl injection volume), von Frey fibers, thermal radiation instrument (specifically for Hargreaves method), transmission electron microscope, laser confocal microscope, whole-cell patch-clamp system.

[0042] II. Experimental Procedure;

[0043] (a) Preparation of recombinant VZV gE protein;

[0044] 1. The amino acid fragment from position 48 to 135 of the VZV gE protein was selected for gene cloning. After codon optimization of the target gene to be adapted to E. coli, it was constructed into a prokaryotic expression vector containing an N-terminal GST tag to form a recombinant expression plasmid.

[0045] 2. The above recombinant expression plasmid was transformed into competent E. coli cells, and after positive clone screening, the cells were expanded and cultured. The host cells were induced to express the target protein under optimized induction conditions.

[0046] 3. Collect the induced Escherichia coli cells, break them up using physical or chemical methods to release the intracellular recombinant fusion protein, remove cell residue by centrifugation, and obtain a crude extract containing the target protein.

[0047] 4. First, the fusion protein in the crude extract was specifically purified by GST Sepharose affinity chromatography, and then combined with subsequent purification processes to finally achieve a protein purity of >95% (SDS-PAgE detection); the purified protein was in liquid form with a predicted molecular weight of 35.6 kDa.

[0048] 5. The purified recombinant VZV gE protein was prepared in a specific storage buffer containing 50% glycerol, 0.395% Tris HCl, and 0.0292% EDTA. The final protein concentration was adjusted to 1 mg / mL, and the product was obtained after aliquoting.

[0049] (II) Preparation of inactivated recombinant VZV gE protein;

[0050] Take the recombinant VZV gE protein, place it in a metal bath, set the temperature to 98℃, and heat for 10 minutes to completely remove the protein activity, thus preparing inactivated recombinant VZV gE protein, and store it at 4℃ for later use.

[0051] (III) Immunogen preparation and animal inoculation;

[0052] 1. Immunogen preparation: The recombinant VZV gE protein solution was dissolved in cell culture grade PBS buffer to obtain immunogens of different concentration gradients (0.05 ng / μl, 0.5 ng / μl).

[0053] 2. Animal grouping and inoculation: SD rats were randomly divided into a model group (VEP) and a control group (IVEP). In the model group, 40 μl of the corresponding concentration of immunogen was injected once into one paw using a microsyringe; in the control group, an equal amount of inactivated VZV gE protein was injected once into one paw. During injection, it was ensured that the drug was injected into the subcutaneous tissue of the paw, avoiding blood vessels and nerves.

[0054] (iv) Model validation and testing;

[0055] 1. Pain phenotype assessment (behavioral testing):

[0056] Testing time points: Tests were conducted on days 3, 7, 14, 21, 28, 35, 42, and 56 after injection.

[0057] Mechanical pain hypersensitivity test: The middle of the sole of the rat's injection side was stimulated with von Frey fibers, and the 50% paw withdrawal threshold (PWT) was measured according to the standard test procedure, and the test data were recorded.

[0058] Thermal hyperalgesia test: Using a thermal radiation meter (Hargreaves method), the middle of the sole of the rat's injection side was targeted, and the time from the start of thermal radiation to the rat's withdrawal response was measured, i.e., the withdrawal latency (PWL), and the test data were recorded.

[0059] Cold pain hypersensitivity test: The acetone test method was used. An appropriate amount of acetone was drawn with a microsyringe and quickly dripped into the middle of the sole of the rat's paw on the injection side. The cold pain response of the rat was observed and recorded within 1 minute.

[0060] 2. Evaluation of nerve damage;

[0061] Transmission electron microscopy: On day 21 after model establishment (3 weeks after modeling), 3 rats were randomly selected from each group. After anesthesia, the main trunk of the sciatic nerve on the injection side was quickly separated. After treatment with 2.5% glutaraldehyde fixation, 1% osmium tetroxide fixation, gradient dehydration, embedding, ultrathin sectioning, staining, etc., the changes in the myelin sheath structure of nerve fibers were observed by transmission electron microscopy.

[0062] Immunofluorescence assay: Three rats were randomly selected from each group on day 14 (2 weeks) and day 28 (4 weeks) after modeling. After anesthesia, tissues from the dorsal root ganglion (DRG) and plantar skin on the injection side were collected, as well as plantar skin tissue 6 hours after modeling. Frozen sections were prepared, permeabilized, and blocked. Primary antibodies including ATF3 antibody, NF200 antibody, IB4 fluorescent probe, CGRP antibody, PRX antibody, PGP9.5 antibody, and VZV gE antibody were added and incubated overnight at 4°C. Then, secondary fluorescent antibodies were added and incubated at room temperature. After DAPI staining, the colocalization of ATF3, NF200, IB4, and CGRP neurons in the DRG tissue and the expression level of PRX were observed using a laser confocal microscope. Intraepidermal nerve fiber density (IENF) and colocalization of PGP9.5 and VZV gE in the plantar skin tissue were also observed.

[0063] (v) Test results;

[0064] Figure 1 The image shows the results of a mechanical pain hypersensitivity test after a single injection of recombinant VZV gE protein into the sole of the foot of a male rat in the von Frey experiment. Figure 1 The 50% withdrawal threshold (PWT) of all groups was close to 15g, indicating that the rats had consistent mechanical pain sensitivity before the experiment. After injection, the PWT of the control group remained at around 12-15g, without significant hyperalgesia. In the model group, PWT decreased significantly from 4 hours onwards, indicating that recombinant VZV gE protein induced mechanical hyperalgesia in male rats.

[0065] Figure 2 The image shows the results of thermal pain hypersensitivity detection in male rats after a single injection of recombinant VZV gE protein into the sole of their paw using a thermal radiation instrument (Hargreaves method). Figure 2 In the study, the withdrawal latency in all groups was approximately 10–11 seconds, indicating that the rats had consistent thermal pain sensitivity before the experiment. After injection, the withdrawal latency in the control group remained around 10–11 seconds, with no significant thermal pain sensitivity observed. In the model group, the withdrawal latency significantly shortened starting at 4 hours, indicating that the recombinant VZV gE protein induced thermal pain hypersensitivity in male rats.

[0066] Figure 3 The image shows the results of a single injection of recombinant VZV gE protein into the sole of the foot of a male rat in an acetone test, resulting in cold pain hypersensitivity. Figure 3In the study, the cold pain response scores of all groups were close to 1 point, indicating that the cold pain sensitivity of the rats was consistent before the experiment. After injection, the control group scores remained between 0 and 2 points, without significant cold pain sensitivity. The model group showed a significant increase in scores starting from 4 hours, indicating that the recombinant VZV gE protein induced cold pain hypersensitivity in male rats.

[0067] Figure 4 This image shows the changes in myelin sheath structure of nerve fibers in the main trunk of the sciatic nerve after a single injection of recombinant VZV gE protein into the plantar surface of a rat. The upper image shows a low-power microscopic observation of the nerve tissue (scale bar corresponds to a larger field of view), and the lower image shows a transmission electron microscopic observation of the nerve fibers (scale bar is 2 μm, showing the ultrastructure). Figure 4 As can be seen, in the control group, the cells / fibers in the nerve tissue were regularly arranged and morphologically intact, the myelin sheath of the nerve fibers was continuous and dense, and the axons were morphologically normal, with no obvious damage. In the model group, the nerve tissue showed obvious structural disorder, with loosely arranged cells / fibers; the myelin sheath of the nerve fibers was broken, exhibiting typical characteristics of nerve injury. This figure verifies the nerve injury basis of the pain model from a morphological perspective: treatment with recombinant VZV gE protein induced pathological changes in nerve tissue (especially the myelin sheath), which is the structural reason for hyperalgesia, further supporting the effectiveness of the animal model.

[0068] Figure 5 Two and four weeks after a single injection of recombinant VZV gE protein into the rat paw, the colocalization of the neuronal injury markers ATF3 and NF200 in DRG tissues was observed. DAPI (blue) marks cell nuclei, showing cell distribution; ATF3 (red) is a neuronal injury marker (expression increases after neuronal injury); NF200 (green) is a marker of large-diameter myelinated sensory neurons; the merged graph shows the colocalization of ATF3 and NF200. Figure 5 In the control group, regardless of whether the model was established for 2 or 4 weeks, ATF3 showed no significant red signal (only a small amount of background was observed), while the NF200 green signal was evenly distributed, indicating that the neurons in the control group were not significantly damaged. In the model group, after 2 weeks of modeling, ATF3 showed a significant red signal, and it partially overlapped with the NF200 green signal in the Merge plot (co-localization). After 4 weeks of modeling, the red signal of ATF3 further increased, and the co-localization ratio with NF200 was even higher. This indicates that after treatment with recombinant VZV gE protein, myelinated sensory neurons (NF200 positive) in the DRG were damaged, and the damage persisted over time. This figure validates the neuronal damage in the model at the molecular level: the high expression of ATF3 directly proves neuronal damage, and the damage occurs in myelinated neurons responsible for mechanical or thermal pain sensation, explaining the cellular mechanism of hyperalgesia in previous behavioral tests and improving the effectiveness of the model.

[0069] Figure 6 Two and four weeks after a single injection of recombinant VZV gE protein into the rat paw, the colocalization of ATF3 and CGRP neurons, markers of nerve injury, in DRG tissue was investigated. DAPI (blue) marks cell nuclei, showing cell distribution; ATF3 (red) represents a marker of nerve injury; CGRP (green) is a marker of nociceptive sensory neurons (transmitting pain); and the merged plot shows the colocalization relationship between ATF3 and CGRP. Figure 6 In the control group, ATF3 showed no significant red signal at both 2 and 4 weeks, while CGRP showed a uniform distribution of green signal, indicating that nociceptive neurons in the control group were not damaged. In the model group, ATF3 showed a significant red signal 2 weeks after modeling, and partially overlapped with the green signal of CGRP in the Merge plot (co-localization). Four weeks after modeling, the red signal of ATF3 continued to increase, and the co-localization ratio with CGRP further increased; this indicates that treatment with recombinant VZV gE protein damaged the nociceptive neurons (CGRP-positive) responsible for transmitting pain in the DRG, and the damage continued to progress over time. This figure explains the cellular mechanism of hyperalgesia in the model at the level of pain-transmitting neurons: damage to nociceptive neurons (CGRP-positive) (high ATF3 expression) directly leads to abnormal pain signal transmission.

[0070] Figure 7 Two and four weeks after a single injection of recombinant VZV gE protein into the rat paw, the colocalization of ATF3 and IB4 neurons, markers of nerve injury, in DRG tissue was observed. DAPI (blue) marks cell nuclei; ATF3 (red) is a marker of nerve injury; IB4 (green) is a marker of unmyelinated nociceptive sensory neurons (transmitting pain); the merged graph shows the colocalization relationship between ATF3 and IB4. Figure 7 In the control group, no significant red signal was observed in ATF3, while the green signal of IB4 was evenly distributed, indicating that the unmyelinated nociceptive neurons in the control group were undamaged. Two weeks after modeling, significant red signals appeared in ATF3 in the model group, and these signals overlapped with the green signal of IB4 in the Merge plot (co-localization, marked with white arrows). Four weeks after modeling, the red signal of ATF3 continued to increase, and the co-localization ratio with IB4 remained at a high level. This indicates that treatment with recombinant VZV gE protein damaged the unmyelinated nociceptive neurons (IB4-positive) responsible for transmitting pain in the DRG, and this damage persisted. This figure supplements the neuronal damage types in the pain model: not only were myelinated neurons (NF200-positive) damaged, but unmyelinated nociceptive neurons (IB4-positive) were also damaged. The combined damage to neurons and myelin is the key cellular basis for the various types of hyperalgesia in the model, further enriching the evidence for the pathological mechanism of the model.

[0071] Figure 8The expression of the myelin sheath structural protein PRX in rat paw skin was observed 2 and 4 weeks after a single injection of recombinant VZV gE protein into the rat paw. DAPI (blue) was used to label the cell nucleus; PRX (red) was used to represent the myelin sheath structural protein. Figure 8 In the control group, a significant red signal was detected in PRX after modeling, and the myelin sheath structure remained intact, indicating no myelin damage. In the model group, after 2 and 4 weeks of modeling, the red signal in PRX significantly decreased, and the continuous and intact myelin sheath structure disappeared, often exhibiting fragmented myelin sheath structures. This indicates that treatment with recombinant VZV gE protein caused damage to the myelin sheath of neurons in the plantar skin, and this damage persisted. This figure supplements the neuronal damage types in the pain model: not only are neurons damaged, but myelin sheaths are also damaged. This combined damage to both types of neurons is the key cellular basis for the various types of hyperalgesia in the model, further enriching the evidence for the pathological mechanism of the model.

[0072] Figure 9 Four weeks after a single injection of recombinant VZV gE protein into the plantar surface of rats, the expression of the myelin structural protein PRX in DRG tissues was observed. DAPI (blue) marks the cell nucleus; PRX (red) represents the myelin structural protein. Figure 9 In the control group, the red signal in PRX detection was normal. Four weeks after modeling, the red signal in the model group significantly increased, possibly due to abnormal over-repair following myelin sheath injury. This indicates that treatment with recombinant VZV gE protein resulted in damage to the myelin sheath of neurons in the DRG tissue, and this damage persisted. This figure supplements the neuronal damage types in the pain model: not only were neurons damaged, but myelin sheaths were also damaged. This combined damage to both types of neurons is the key cellular basis for the various types of hyperalgesia in the model, further enriching the evidence for the model's pathological mechanism.

[0073] Figure 10 shows the results of related tests on the plantar skin of rats 6 hours, 2 weeks, and 4 weeks after a single injection of recombinant VZV gE protein. (a) shows the statistical results of the morphology of the nerve fiber marker PGP9.5 and the intradermal nerve fiber density (IENF), and (b) shows the co-localization results of the nerve fiber marker PGP9.5 and VZV gE protein. DAPI (blue) marks the cell nucleus; PGP9.5 (red) is a broad-spectrum nerve fiber marker; VZV-gE (green) is the VZV viral gE protein; and the merged graph shows the co-localization relationship between PGP9.5 and VZV-gE. Figure 11Detailed images showing the colocalization of PGP9.5, a broad-spectrum nerve fiber marker, with VZV gE protein in rat plantar skin at 6 hours, 2 weeks, and 4 weeks after a single injection of recombinant VZV gE protein. The control group had approximately 7 small nerve fibers per 200 μm in the plantar skin. Statistical analysis of the model group showed no statistically significant difference in small nerve fiber density between the model group and the control group at 6 hours. However, the model group showed a decrease in small nerve fiber density compared to the control group at 2 and 4 weeks; this indicates that treatment with recombinant VZV gE protein damaged the small nerve fibers in the plantar skin. Figure 11 In the control group, no obvious green signal was observed in VZV-gE after modeling, indicating that the control group's plantar skin lacked VZV viral gE protein. In the model group, 6 hours after modeling, VZV-gE showed a significant green signal and co-localized with PGP9.5-labeled nerve fibers. At 2 and 4 weeks after modeling, the green signal of VZV-gE was significantly reduced compared to 6 hours, but it was still detectable and co-localized with PGP9.5-labeled nerve fibers. This indicates that after treatment with recombinant VZV gE protein, VZV gE protein is involved in the damage of small nerve fibers in the plantar skin during both the acute and chronic phases.

[0074] Figure 12 KEGG enrichment analysis of rat foot skin tissue transcriptome sequencing 3 weeks after a single injection of recombinant VZV gE protein into the foot. Figure 13 KEGG enrichment analysis of DRG tissue transcriptome sequencing 3 weeks after a single injection of recombinant VZV gE protein into the rat paw. Figure 12 It can be clearly seen that key signaling pathways related to postherpetic neuralgia (such as neuroinflammation, cell damage, immune response, and nerve repair / remodeling pathways) in the plantar skin tissue are significantly activated or inhibited, reflecting the molecular pathological changes induced by VZV gE protein in the local skin. It also reveals that the gene expression profile in the plantar skin undergoes adaptive changes when it is used as the injection site for modeling. These pathway changes are directly related to skin nerve fiber damage and the formation of local inflammatory microenvironment, and are the molecular basis of the local pathophysiology of the skin in postherpetic neuralgia. Figure 13 The study clearly identified signaling pathways significantly enriched by VZV gE protein in DRG tissue, mainly concentrated in areas such as nerve damage repair, neuroinflammation, pain signal transduction, and neuronal apoptosis / survival. This reflects the specific molecular response of DRG neurons in neuropathic pain states and reveals that gene expression in the peripheral pain center (DRG) is reprogrammed during herpes zoster neuralgia. These pathway changes are the core molecular mechanisms of neuronal damage and abnormal amplification of pain signals (hyperalgesia / hyperalgesia), explaining the peripheral nerve regulation basis of persistent neuralgia in the model. Figure 11 and Figure 12This study clarifies the correlation between local skin damage and molecular changes in DRG ganglia, providing dual molecular evidence from both peripheral nerves and target tissues for a comprehensive understanding of the pathogenesis of herpes zoster neuralgia. It also provides pathway and molecular target references for the development of analgesics targeting DRG.

[0075] The behavioral test results above showed that, starting from day 3 after injection, the model group rats exhibited significant mechanical hyperalgesia (decreased PWT), thermal hyperalgesia (shortened PWL), and cold hyperalgesia (enhanced cold pain response), and this pain phenotype persisted until day 56 without recovery, showing a significant difference from the control group. Transmission electron microscopy revealed sciatic nerve myelin sheath rupture and other damage in the model group rats, while the myelin sheath structure remained intact in the control group. Immunofluorescence assays showed that the colocalization rate of ATF3, NF200, IB4, and CGRP neurons in the DRG tissue of the model group was significantly higher than that in the control group; PRX expression in the plantar skin and DRG tissue of the model group was significantly higher than that in the control group; and the density of small nerves in the plantar skin of the model group was significantly lower than that in the control group. Based on these results, it can be confirmed that this embodiment can construct a rat model of herpes zoster neuralgia.

[0076] Example 2: Application of an animal model of herpes zoster neuralgia in drug screening.

[0077] Using the rat model of herpes zoster neuralgia constructed in Example 1, the efficacy of a candidate analgesic drug (e.g., a novel neuroinflammation inhibitor) was evaluated.

[0078] Rats that successfully developed the model were randomly divided into a model control group and a drug administration group.

[0079] The drug administration group was given the candidate drug by gavage or intraperitoneal injection, while the model control group was given an equal amount of solvent.

[0080] Before and after drug administration, the mechanical pain threshold and thermal pain latency of rats in each group were detected according to the method in Example 1.

[0081] Results analysis: If the pain behaviors (such as increased PWT and prolonged PWL) of rats in the drug administration group are significantly improved compared with those in the model control group, it indicates that the candidate drug has the potential to treat herpes zoster neuralgia.

[0082] Example 3: Study on the pathogenesis of herpes zoster neuralgia

[0083] Using the herpes zoster neuralgia animal model of Example 1, we explored the key molecular changes and pathophysiological mechanisms related to the nervous and immune systems during the pathogenesis of herpes zoster neuralgia.

[0084] A rat model of herpes zoster neuralgia and age-matched healthy rats (as normal control group) were used.

[0085] At different key time points after modeling, rats were randomly selected from the model group and the normal control group. After anesthesia, dorsal root ganglia, sciatic nerve, spinal cord and related immune tissues were collected. Some samples were used for histological observation and some samples were used for molecular level detection.

[0086] Rats were assessed for pain-related behaviors using standard methods, and phenotypic changes such as hyperalgesia and allergy were recorded.

[0087] The structure of nerve fibers was observed using transmission electron microscopy, and the morphology and distribution of nerve cells and immune cells were analyzed using laser confocal microscopy.

[0088] Western blot and real-time quantitative PCR were used to detect the expression levels and gene transcription of neuroinjury markers, inflammatory factors, key proteins in signaling pathways, and other methods; immunohistochemistry was used to clarify the tissue localization of target molecules.

[0089] Based on the above detection results, the association between differentially expressed molecules and pain phenotypes and nerve damage was analyzed to preliminarily screen key pathways or molecules involved in the pathogenesis. Their roles can be further verified through in vitro cell experiments. Furthermore, by comparing the detection data of the model group and the normal control group, if the model group shows abnormal expression of specific molecules, damage to neural structures, and changes in pain behavior, and if these three are logically related, the role of that molecule or related pathway in the pathogenesis of herpes zoster neuralgia can be revealed, providing experimental support for elucidating the disease's pathogenesis.

[0090] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for constructing an animal model of herpes zoster neuralgia using recombinant VZV gE protein, characterized in that: Includes the following steps: Step 1: Prepare an immunogen from recombinant VZV gE protein; the recombinant VZV gE protein is expressed in Escherichia coli and then purified. Step 2: Inject the immunogen into a non-human mammal to induce persistent pain behavior in the non-human mammal, thereby constructing an animal model of herpes zoster neuralgia.

2. The method for constructing an animal model of herpes zoster neuralgia using recombinant VZV gE protein according to claim 1, characterized in that: The non-human mammal in question is a rat.

3. The method for constructing an animal model of herpes zoster neuralgia using recombinant VZV gE protein according to claim 1, characterized in that: The concentration of the recombinant VZV gE protein in the immunogen was from 0.05 ng / μl to 0.5 ng / μl.

4. The method for constructing an animal model of herpes zoster neuralgia using recombinant VZV gE protein according to claim 3, characterized in that: The single injection dose of the immunogen is 30-50 μl.

5. The method for constructing an animal model of herpes zoster neuralgia using recombinant VZV gE protein according to claim 1, characterized in that: The injection was a subcutaneous injection into the sole of the foot.

6. The method for constructing an animal model of herpes zoster neuralgia using recombinant VZV gE protein according to claim 1, characterized in that: The successful establishment of the herpes zoster neuralgia animal model was confirmed by detecting pain-related behaviors in animals using behavioral methods and by examining nerve damage in animals using immunofluorescence and transmission electron microscopy. The behavioral methods include one or more of mechanical hyperalgesia, thermal hyperalgesia, and cold hyperalgesia.

7. An animal model of herpes zoster neuralgia constructed by the method of any one of claims 1 to 6.

8. The use of the animal model of herpes zoster neuralgia according to claim 7 in screening or evaluating drugs for the prevention or treatment of herpes zoster neuralgia.

9. The application of the animal model of herpes zoster neuralgia according to claim 8 in the study of the pathogenesis of herpes zoster neuralgia.

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